Migration is a cellular process important for many physiological functions. During development, cells rearrange to form structured tissues and organs, and in the adult, leukocytes exit the blood stream and move into tissue in response to signals reporting the presence of foreign invaders. In cancer, metastases are formed by migrating cancer cells. In the first two examples above, migration is directional toward a specific target. This behavior is known a chemotaxis and is brought about by a gradient of chemoattractants or chemokines that migrating cells are able to sense and orient their polarization (i.e. axis of migration) accordingly. Some cells migrate randomly in the absence of chemokine gradients. Cytokines may cause those cells to migrate at a faster pace, although still randomly, a process known as cytokinesis.
Many different methods have been developed to study cell migration. Choosing between those methods generally involves a trade-off between throughput and information content. On one end of the spectrum lies the Dunn chamber in which a gradient is formed between a circular chamber and a concentric annular chamber etched in glass. A coverslip with cells growing on it is then inverted on top of the chambers and their migration in the vicinity of the annular bridge between the chambers is recorded using video-microscopy. Later (i.e. off-line) individual cells are identified manually (sometimes semi-automatically) and their position tracked over time. The Dunn chamber is very low throughput but yields precise information about individual cell behavior. Such detail is important for studies of the fundamental mechanisms of cell migration but may not be practical for studying the effect of numerous conditions, such as in anti-inflammatory drug screening. On the opposite end of the spectrum we have microtiter plate-insert transwell assays, wherein cells are seeded on a porous membrane suspended in a microtiterplate and a chemoattractant is introduced on the opposite side of the membrane. The chemoattractant diffuses through the membrane forming a gradient. A typical readout entails removal of the cells on the side where they were seeded and quantification of the cell number on the other side. As an example, cells can be manually scraped from the original side so that the transwell insert only contains cells that have traversed the membrane. Alternatively, only cells that fall from the membrane to the bottom of the well below may be included in the readout. The cells may be quantified by cell lysis and an Adenosine Triphosphate (ATP) measurement via bioluminescence which can be detected using a plate reader. In the case of transwell assays, the steepness of the gradient is hard to determine, and no distinction is made between chemokinesis, chemotaxis, or even cell death. Additionally, the inserts themselves are expensive and the specific steps of the assay, namely, the removal of non-migrating cells and the washing of cells on the underside of the membrane, are non-trivial to automate.
As is known, microtechnology has been applied to make precise soluble gradients with a variety of spatial and temporal profiles, as well as, surface-bound gradients. Microfluidic gradient generation devices have been employed to study the migration of blood cells, cancer cells, neurons, as well as, bacteria. However, assays that seed cells randomly in a chamber and employ laminar flow to create a gradient rely on video-microscopy for readout, and thus, limits throughput and requires expensive software for data analysis. Additionally, the presence of flow may eliminate potential paracrine signals that are involved in the biological process of interest. For example, if a drug is to be developed that affects tissue resident cells that produce the stimulus for white blood cell migration into tissue, the presence of flow in the screening assay would abolish this signal and lead to the discovery of drugs that treat the symptom rather than the root of the problem.
Therefore, it is a primary object and feature of the present invention to provide a method for quantifying cell motility and cell migration.
It is a further object and feature of the present invention to provide a method for studying cell migration that allows a user to simply and easily determine the quantitative motility and directional migration data for a cell population.
It is a still further object and feature of the present invention to provide a method for studying cell migration that combines microfluidic gradient generation with micro-patterning to simplify the extraction of important migratory information.
In accordance with the present invention, a method of quantifying cell migration of a cell population is provided. The method includes the steps of patterning the cell population within a channel network through a first body and obtaining an first image of the cell population. A second image of the cell population is obtained after a first predetermined time period. Thereafter, the first and second images are compared.
The channel is defined by a first sidewall and the method contemplates the additional step of providing a first predetermined medium along the first sidewall. In addition, may be defined by a second sidewall. A second predetermined medium may be provided along the second sidewall. Alternatively, a predetermined medium may be provided in a second channel through the first body. The second channel is interconnected to the first channel. The first channel may include an input and an output, and the second channel includes an input and an output communicating with the output of the first channel.
The channel network is defined by a first sidewall and the method may include the additional step of providing a first predetermined medium along the first sidewall. In addition, the channel network may be defined by a second sidewall and the method may include the additional step of providing a second predetermined medium along the second sidewall.
The channel network may include first and second interconnected channels. A predetermined medium may be provided in the first and second channels. The first channel includes an input and an output and the second channel includes an input and an output communicating with the output of the first channel. The first and second channels communicate along a cross channel. The first channel has a first cross-sectional area and the second channel has a second cross-sectional area. The cross channel has a cross-sectional area smaller than the cross-sectional areas of the first and second channels.
The method may include the additional steps of patterning a second cell population within a channel network through a second body and obtaining an image of the second cell population after the first predetermined time period. The image of the second cell population is compared with the second image of the first cell population.
The step of comparing may includes the additional steps of calculating a first center of gravity for the cell population at a first initial time and calculating a second center of gravity for the cell population after the predetermined time period. The absolute difference between the first and second centers of gravity is a quantitative measure of the average directional migration of the cells population. In addition, a first full-width at half mass for the cell population is calculated at a first initial time and at after a predetermined time period. The absolute difference between the first full-width at half mass and the second full-width at half mass is a quantitative measure of the average motility of the cell population. Other comparable measures can be envisioned, such as the parameters acquired via curve-fitting of the cell distribution using known distribution functions such as the Gaussian distribution. In the case of the Gaussian distribution the center of mass and full width at half mass would be replaced by the mean (position) and the variance respectively.
In accordance with a further aspect of the present invention, a method of quantifying cell migration of a cell population is provided. The method includes the step of patterning the cell population within a channel network in a first body. The cell population is observed at a first predetermined time period and at a second predetermined time period.
The method includes the additional of step of providing a first predetermined medium in communication with the channel network. The channel network is defined by first and second sidewalls. The step of providing a first predetermined medium in communication with the channel network includes the additional step of patterning the predetermined medium along at least one of the first and second sidewalls.
The channel network includes first and second interconnected channels. The first and second channels are interconnected by a cross-channel. The first channel has a first cross-sectional area and the second channel has a second cross-sectional area. The cross channel has a cross-sectional area smaller than the cross-sectional areas of the first and second channels.
The step of observing the cell population at a second predetermined time period includes the steps of calculating a quantitative measure of the average directional migration of the cells population and calculating a quantitative measure of the average motility of the cell population. This is accomplished by calculating a first center of gravity for the cell population at a first initial time and calculating a second center of gravity for the cell population after the predetermined time period. The absolute difference between the first and second centers of gravity is a quantitative measure of the average directional migration of the cells population. In addition, a first full-width at half mass for the cell population is calculated at a first initial time and after the predetermined time period. The absolute difference between the first full-width at half mass and the second full-width at half mass is a quantitative measure of the average motility of the cell population.
In accordance with a still further aspect of the present invention, a method is provided for quantifying cell migration of a cell population. The method includes the step of patterning the cell population within a channel network through a first body. A predetermined medium in communication with the first channel is provided. Thereafter, migration of the cell population is observed over a predetermined time period.
The channel network is defined by first and second sidewalls and the step of providing the predetermined medium in communication with the channel network includes the additional step of patterning the predetermined medium along the first and second sidewalls. The channel network may include first and second channels. The first and second channels are interconnected by a cross-channel. The first channel has a first cross-sectional area and the second channel has a second cross-sectional area. The cross channel has a cross-sectional area smaller than the cross-sectional areas of the first and second channels.
The step of observing the cell population at a second predetermined time period includes the steps of calculating a quantitative measure of the average directional migration of the cells population and calculating a quantitative measure of the average motility of the cell population. This is accomplished by calculating a first center of gravity for the cell population at a first initial time and calculating a second center of gravity for the cell population after the predetermined time period. The absolute difference between the first and second center of gravities is a quantitative measure of the average directional migration of the cells population. In addition, a first full-width at half mass for the cell population is calculated at a first initial time and after the predetermined time period. The absolute difference between the first full-width at half mass and the second full-width at half mass is a quantitative measure of the average motility of the cell population.